LAND APPLICATION OF SEWAGE XA9745799 SLUDGE: PATHOGEN ISSUES
A.C. CHANG Department of Soil and Environmental Sciences, University of California at Riverside, Riverside, California, United States of America
Abstract
Diseases transmitted via die faecal-oral exposure route cause severe gastroenteric disorders, and large numbers of causative organisms are discharged with the faecal matter of infected individuals. For this reason, pathogenic bacteria, viruses, protozoa, or helminths, are always found in sewage sludge. If not properly treated for use in agriculture, sludge can be a source of pathogenic contamination. Radiation is an attractive method to reduce (he numbers of microorganisms in sewage sludge. Routine examination for pathogens is not practised nor recommended because complicated and costly procedures are involved. Instead, an indicator organism is usually assayed and enumerated. In this paper, methods are discussed for the investigation of pathogens in sewage sludge.
1. INTRODUCTION
Diseases of sanitary significance are transmitted via the faecal-oral exposure route by which causative agents are orally ingested, rapidly multiply in the intestinal tract causing severe gastroenteric disorders, and are discharged in large numbers with the faecal matter. Even after an infected individual has recovered from the illness, he/she may be a carrier of the pathogen for a long time. For this reason, pathogens - bacteria, viruses, protozoa, or helminths - are always found in domestic sewage and solid residuals of the treatment process, the sewage sludge. If these pathogens are not adequately separated, properly treated and disposed of, sewage and sewage sludge often become the sources of disease. With the frequency and volume of international travel nowadays, pathogens causing outbreaks of disease in one part of the world may appear in the wastewater of another part of the world in a very short time-span. The pathogens found in domestic sewage and sewage sludge are numerous (Table I). The types present vary from commumty to community depending on prevalence of particular diseases among residents, population density, nature of the wastewater collection system, and season.
2. DETECTION AND ENUMERATION OF PATHOGENS
No single procedure is available to isolate, identify, and enumerate all pathogens simulaneously. To estimate the disease-causing potential of a sample, each pathogenic species must first be separated and identity confirmed using biochemical and/or microscopic techniques, and then enumerated. Microbiological Examination in Standard Methods for the Examination of Water and Wastewater, Part 9000 [1] outlines general procedures used by the water and wastewater profession
183 TABLE I. PATHOGENS IN SEWAGE AND SLUDGE
Group Pathogen Disease
Bacteria Salmonella (> 1700 strains) Typhoid fever, salmonellosis Shigella spp. (4 strains) Bacillary dysentery Enteropathogenic E. coli Gastroenteritis Yersinia entericolitica Gastroenteritis Campylobacter jejuni Gastroenteritis Vibrio cholerae Cholera Leptospira Weil's disease Protozoa Entamoeba histolytica Dysentery, colonoid ulcération Giardia lamblia Diarrhea Balantidium coli Diarrhea, colonoid ulcération Cryptosporidium spp. Cryptosporosis Helminths Ascaris lumbricoides Ascariasis (round worm) Ancyclostoma duodenale (Hock worm) Necator americanus (Hook worm) Taenia saginata Taeniasis (Tape worm) Virus Enteroviruses (strains)
Poliovirus (3) Meningitis, paralysis, fever Echovirus (31) Meningitis, diarrhea, rash Hepatitis Type A Infectious hepatitis Coxsackivirus (33) Meningitis, respiratory disease
Norwalk virus Diarrhea, vomiting, fever Calicivirus Gastroenteritis Astrovirus Gastroenteritis Reovirus (3) Respiratory disease Rotavirus (2) Diarrhea, vomiting Adenovirus (40) Respiratory disease
for assaying the pathogens listed in Table I. Routine examination of water and wastewater for pathogens, however, is not practised nor recommended, because such examinations involve complicated procedures, require trained and experienced personnel and specialized laboratory facilities, and they are time consuming and costly. Instead, an indicator organism is assayed and enumerated. Indicator organisms and pathogens inhabit the intestinal tract of warm-blooded animals and both types are present in faecal discharge. The ideal indicator organism has the following characteristics. • It is present when pathogens are present, for example in sewage sludge, and always absent when pathogens are absent. • Its density in the medium has a direct and constant relationship to pathogen densities.
184 • It is unable to reproduce in the media it contaminates. • It survives in the foreign environment for at least as long as the pathogens. As the indicator organism may be detected, isolated, and enumerated by simple, rapid, and economical assay-procedures, it is used routinely to indicate recent contamination by faecal discharge from warm- blooded animals, potential presence of pathogens, and efficiencies of water- and wastewater-treatment processes. Faecal coliforms are most commonly used as indicator organisms. They are present in the gut and faeces of warm-blooded animals in large numbers, and are capable of producing gas from lactose in suitable culturing media at 44.5±0.2°C. Coliforms from other sources often are not capable of producing gas under these conditions, therefore this criterion is used to isolate the faecal component of the group. The survival characteristics of faecal coliforms and of some bacterial pathogens are similar in a water environment and during disinfection, whereas viruses, protozoon cysts, and helminth ova usually are more resistant to adverse conditions and frequently have greater longevity. Although direct and constant relationships between densities of faecal coliforms and pathogens do not always hold, experience has established the significance of coliform group density as a measurement of contamination and, therefore, of sanitary quality of water and wastewater. With sewage sludge and sludge-treated soils, solids interfere with microbial determinations and it is essential that they are removed before assay. Procedures for handling high-solid-content samples are by no means standardized. Samples of low pathogen density are especially difficult, as the volume of sample required for a meaningful determination may be large.
3. PATHOGEN SURVIVAL
3.1. Irradiated sewage sludge Experiments in irradiating sewage sludge were first done more than 40 years ago. Radiation technology has steadily advanced and has found application in disinfecting medical equipment and food, but it has not been widely adopted for wastewater treatment. The process involves exposing the material to y-radiation to reduce its pathogen density. Cesium-137 and cobalt-60 are commonly used and are logical sources for such purposes. Alternatively, accelerated electrons may be employed for disinfection of sewage sludge. Gamma radiation induces ionization in biological tissues resulting in the production of free radicals that cause denaturation of cell protoplasm. Membranes and cell walls may also be damaged, causing lysis. Most pathogens are single-cell organisms that are inactivated when cell protoplasm is damaged. In sewage sludge, pathogens account for a minute fraction of the mass, and are scattered throughout the entire volume of material. Unless every pathogen is exposed to the radiation and the absorbed dosage is adequate, the inactivation will not be effective. Mathematically, the reduction of pathogens in sewage sludge by radiation is a function of the absorbed dose and may be described by a first-order reaction equation. The dosage of ionized radiation required for inactivation appears to vary with pathogen type and the moisture content of the sewage sludge. Salmonella species have D10 values as low as 0.25 kGy in sewage sludge (Table II; D10 refers to the radiation dose (kGy) required for a 90% reduction in organism density). However, the absorbed dosage would rise to almost 1 kGy for dried sludge if a 90% reduction of density were needed. Greater ionizing radiation doses are required to inactivate parasite ova and viruses. Early investigators used as much as 10 kGy absorbed dosage to ensure inactivation of naturally occurring
185 TABLE II. RADIATION ABSORBED DOSAGE FOR IN ACTIVATION OF SELECTED BACTERIA IN SEWAGE SLUDGE [2]
Organism Liquid Composted Dried (2-10% solids) (40% solids) (90% solids) (kGy) (kGy) (kGy)
Coliform group 0.2-0.3' 0.2-0.3 0.15-3.5 Salmonella spp. 0.25-0.5 0.3 0.35-0.8 Faecal Streptococcus 1.2-1.5 1.2-1.5 0.7-3.6 Mycobacter 1.6-3.0 _
*D,0 values: absorbed dose required for each 90% reduction m bactenalcounts.
Ascaris ova in the filter cake of anaerobically digested sludge and in composted sludge [2]. The required dosage for an effective reduction of Ascaris ova is probably considerably less than 10 kGy. Other investigations showed that 1-1.5 kGy absorbed dose resulted in at least two orders of magnitude reduction in Ascaris ova in sewage sludge [3]. Horak [4] found that the viability of Ascaris ova started to decrease at a radiation absorbed dose of 0.5 kGy and no viable ova were observed at dose > 1.1 kGy. Although viruses are susceptible to adverse conditions in the environment such as high temperature, desiccation, and the presence of small amounts of ammonia (such as in anaerobically digested sludge), they have relatively high resistance to inactivation by ionizing radiation. Data in the literature reported a DJ0 of 2.5 kGy for inactivation of viruses in sewage sludge. Due to the difficulty of recovering viruses from sewage sludge, such inactivation studies always involve tracking a large amount of virus that has been artificially introduced into the medium being investigated. The actual dose required to reduce a small amount of viruses in sewage sludge to an acceptable level probably is lower. In the opinion of radiation scientists, 3-5 kGy of ionized radiation is adequate to completely inactivate pathogens in sewage sludge [5].
3.2. Sludge-treated soils The soil is a hostile environment for pathogens that inhabit the intestinal tract of warm-blooded animals. They fail to multiply and rapidly die off due to adverse conditions and microbial antagonism. Generally, unfavorable conditions for survival of pathogens in soils are: desiccation, high ambient temperature, acidic pH, and low organic matter content. Published data show die-off rate constants
(number of days for log10 reduction in organism density) for indicator organisms, bacterial pathogens, and viruses, ranging from 0.04 to < 10 [6]. At these rates, pathogens introduced in sewage sludge may be reduced to non-detectable levels in a 100- to 150-day crop-growing season (Table III) [7]. Few data are available on survival of protozoon cysts and helminth ova. For sludge deposited on the soil surface and not incorporated, a 30-day period is considered adequate to eliminate the hazards of transmitting parasitic diseases [8]. Because of their ability to resist adverse environmental conditions, cysts and ova incorporated into the soil may survive for extended periods of time (Table III). Radiation reduces pathogen density in sewage sludge significantly, if not altogether. Any remaining pathogens would be expected to be weakened and to die off rapidly in the soil.
186 TABLE HI. REPORTED PATHOGEN SURVIVAL TIME IN SOILS [7]
Organism Time (days)
Coliform Group 38 Faecal Streptococcus 26-77 Salmonellae spp. 15 - > 180 Salmonella typhi 1- 120 Entomoeba histotytica cysts 6-8 Ascaris ova up to 7 years Hook larvae 42 Enterovirus 8- 175
4. PROTOCOLS FOR PATHOGEN-SURVIVAL INVESTIGATIONS
The effectiveness of ionizing radiation for disinfecting sewage sludge was demonstrated in the IAEA Co-ordinated Research Programme (CRP) (E3-40.03) on Radiation Treatment of Sewage Sludge for Safe Realization (May 1983 - July 1990). As for pathogen survival in the current CRP (Dl- 50.04) on The Use of Irradiated Sewage Sludge to Increase Soil Fertility, Crop Yields and to Preserve the Environment, initiated in July 1995, the objectives should be limited to: • Compare the pathogen characteristics of irradiated and non-irradiated sewage sludges used in field experiments. • Demonstrate that no pathogen accumulated in soil through repeated applications of irradiated sewage sludge. Pathogen assays, however, require specialized laboratory facilities and a trained technician. As none of the participants of the current CRP has experience or is equipped to undertake pathogen determination, this part of the study should be kept to a minimum and be as simple as possible. Instead of attempting comprehensive pathogen determinations, participants should utilize local facilities where exist the necessary capability and expertise. Usually, public-health groups in universities (medical schools) and hospitals have access to laboratories that routinely perform pathogen assays. Participants should seek expert advice if they plan to conduct this phase of the work in their own laboratories.
4.1. Sampling procedures Each participant is required to determine the density of faecal coliforms and Ascaris ova in sewage sludge to be applied to soil. The requirements are outlined as follows.
4.1.1. Sewage sludge Because sewage sludge undergoes further changes following radiation, samples should not be obtained immediately following the irradiation process. Instead, samples should be obtained at the time of application to the experimental plots. One sample should be taken from the material going to each
187 experimental plot. As non-irradiated sewage sludge is also used in the experiment, comparable non- irradiated samples should be obtained. According to the previously agreed experimental design, there will be a total of 40 samples (note: the number of samples will vary depending on the number of treatments used) accounting for four replications of: • control (no sewage sludge + 15N labeled fertilizer at locally recommended rate, 1% a.e.), • sewage sludge, 50% of recommended N rate • sewage sludge, 100% of recommended N rate, • sewage sludge, 150% of recommended N rate, and • sewage sludge, 200% of recommended N rate for both irradiated sludge and non-irradiated sludge treatments. Each sample should consist of approximately 500 g (in dry weight equivalent) if the material is in solid form or 500 mL if the material is in liquid form obtained from a composite of random grab samples. Samples should be refrigerated or stored in ice-packed coolers immediately and the pathogen determination should begin as soon as the samples are received at the laboratory. Noticeable changes in organism density and types have been reported in unrefrigerated samples, especially when the ambient temperature is > 13°C. Samples of field moisture content are used in pathogen determination and no drying is needed. During the sampling, extraordinary caution should be exercised to prevent cross-contamination between irradiated and non-irradiated sludges (do not use the same sampling tool for irradiated and non-irradiated sludges, or sample the irradiated sludges first; transport and store irradiated and non-irradiated sludge samples separately; alert the personnel involved in handling samples and in determining pathogens of the need for separating these samples). It is also advisable that the sample taker should avoid direct contact with any sewage sludge and wash hands after handling the samples. The pathogen determination for sewage sludges will be done only once during the entire 5-year experimental period.
4.1.2. Sludge-treated soils Soil from plots receiving irradiated and non-irradiated sewage sludge will be sampled after the last crop of the experiment is harvested. This one-time sampling will involve only the experimental plots corresponding to the sewage-sludge sampling. The soil will be sampled from the surface to the depth of sludge incorporation. The general procedures outlined in the previous section for obtaining and handling the sludge samples should be followed for soil sampling.
5. PATHOGEN ASSAYS
All of the samples used for pathogen analysis should be sieved to pass through a 2-mm screen to insure homogeneity of the sub-samples drawn for the determinations.
5.1. Faecal coliforms The faecal coliform determination starts with an aliquot of approximately 64 g (dry weight equivalent) of sludge solids or 64 mL of liquid sludge. Each aliquot will be mixed and divided into quarters. One of the quarters will be selected for further sample subdivision and the remainder may be discarded. The sludge aliquot will be divided in this manner three times until approximately 1 g (dry weight equivalent) or 1 mL of sample is obtained.
188 The sample (1 g or 1 mL) is then diluted to a volume of 100 mL using sterile water, and appropriate series dilutions made for faecal coliform determinations. To avoid complications in filtering and developing a bacterial colony from high-solid suspensions, the multiple-tube fermentation technique should be used for the assay. The procedures for preparing the culture media, incubation, and enumeration may be found in Multiple-tube Fermentation Technique for the Coliform Group (Part 9221) in the Standard Methods for the Examination of Water and Wastewater [1]. The detection limit of this procedure is approximately 2 MPN per 1 g or 1 mL of sewage sludge. (MPN stands for most probable number, in this case of faecal coliforms, in a given unit of sample; it is a statistically derived estimation of cell density.) The data should be adjusted according to moisture content of the sludge and reported as MPN of faecal coliform per g sludge or soil dry weight.
5.2. Ascaris ova This part of the assay starts with 320 g (dry weight equivalent) of solid sludge or 320 mL of liquid sludge. Using the previously described quartering technique, the material is divided three times to obtain a sample of approximately 5 g or 5 mL. If the sample is liquid, it is centrifuged at 2000 rpm for 5 min and the supernate discarded. The Ascaris ova will be recovered from the solids through successive flotation (the ova will float on the surface) using saturated NaNO3 solution (density = 1.38g/mL). The recovered ova will then be incubated in Petri dishes with saline containing 0.8% formaldehyde for a period of 8 weeks. At the end of the incubation, viable ova (those able to embryonate) will be counted. The detectability of this procedure is approximately 0.2 ova /g or /mL. Standardized procedures for isolation and enumeration of helminth ova in sewage and sewage sludge are not available. Textbooks on helminthology should be consulted for specifics in procedures for identifying ova and judging embryonation.
6. RECOMMENDATIONS
The results of pathogen determinations are procedure-dependent. For consistency of data and comparisons between sites, it is advisable that all participants follow the same general procedures and make only minor modifications to suit special local conditions. There are no data in the technical literature on pathogens in irradiated sludge-treated soils. It is possible that data from this CRP on "The Use of Irradiated Sewage Sludge to Increase Soil Fertility, Crop Yields and to Preserve the Environment" may be pooled and published in the future.
REFERENCES
[1] ANONYMOUS, Standard Methods for the Examination of Water and Wastewater, 18th edition. American Public Health Association, (1992) New York. [2] BRANDON, 1979. "Pathogen reduction in sludges by irradiation", Sandia Irradiator for Dried Sewage Solids, Seminar Proceedings and Dedication, October 18-19, 1978, Albuquerque, New Mexico, Sandia Laboratory Energy Report Sandia, 79-0182, Applied Biology and Isotope Utilization Division, Sandia National Laboratory (1979) 37-47, [3] YEAGER, J. G., O'BRIEN, R.T., Irradiation as a means to minimize public health risks from sludge-borne pathogens, J. Water Pollut. Contrl. Fed. 55 (1983) 977-983. [4] HORAK, P., Experimental destruction of Ascaris ova in sewage sludge by accelerated electron, Irradiation Water Research 28 (1994) 939-941.
189 [5] PIKAEV, A. V., "Current Status of Radiation Treatment of Water and Wastewater", Sewage and Wastewater for Use in Agriculture, IAEA TECDOC (1997) (this volume). [6] GERBA, C. P., "Pathogens," Proc. 1983 Workshop, Utilization of Municipal Wastewater and Sludge on Land (PAGE, A.L., GLEASON, T.L., SMITH, J.E. (Jr.) ISKANDAR, I.K., SOMMERS, L.E., Eds.) University of California, Riverside, (1983) 460 pp. [7] FRANKENBERGER W. T. (Jr.), "Fateof wastewater constituents in soil and groundwater: Pathogens", Irrigation with Reclaimed Municipal Wastewater, A Guidance Manual (PETTYGROVE, G.S., ASANO, T., Eds.) California State Water Resources Control Board, Report No. 84-1 (1984). [8] U.S. ENVIRONMENTAL PROTECTION AGENCY, Standards for the use or disposal of sewage sludge final rules. Federal Register 58 (1993) 9248-9415.
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